The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates.

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters

Effect of entolimod treatment on G-CSF and IL-6 levels in peripheral blood of irradiated NHPs.

<p><b>A, B</b>: Effect of different entolimod doses administered 1 h after LD_50/40 TBI (6.75 Gy; study Rs-09; N = 18). <b>C, D</b>: Effect of different entolimod doses administered 25 h after LD_50/40 TBI (6.75 Gy; study Rs-14; N = 10). <b>E, F</b>: Comparison of dose-dependence of background-adjusted Area Under the Curve (AUC_0-24) values for G-CSF and IL-6 after entolimod treatment given 1 h versus 25 h after LD_50/40 TBI (with dashed log-linear regression lines). Error bars represent standard errors.</p

Enhanced morphological recovery of hematopoietic and lymphoid organs in NHPs treated with entolimod post-irradiation.

<p>NHPs were treated with a single injection of 40 μg/kg entolimod 16, 25 or 48 hours after LD_75/40 total body irradiation (TBI). Tissue morphology was assessed 40 days post-irradiation and compared to that in control NHPs treated with vehicle 16 hours after LD_75/40 TBI. Representative histological images (hematoxylin-eosin staining) of sternum bone marrow sections, thymuses, spleens and mesenteric lymph nodes of animals that survived to study termination on Day 40 post-TBI (study Rs-06) are shown. Scale bars: 100 μm for bone marrow, 200 μm for thymus, spleen, and lymph node.</p

Histological evaluation of hematopoietic/lymphoid organs from NHPs that survived to day 40 after 6.5 Gy TBI and vehicle or entolimod treatment (study Rs-06)

<p>^A Scoring was performed based on a 5-grade scale developed for each organ: 0 –total aplasia; 1 –pronounced atrophy, 2 –moderate atrophy, 3 –slight atrophy, close to normal morphology; 4 –normal morphology. Scoring criteria for individual organs are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135388#pone.0135388.s008" target="_blank">S1 Methods</a>.</p><p>^B Student’s t-test vs. vehicle, 2-tailed.</p><p>Histological evaluation of hematopoietic/lymphoid organs from NHPs that survived to day 40 after 6.5 Gy TBI and vehicle or entolimod treatment (study Rs-06)</p

Efficacy of a single injection of entolimod in increasing 40-day survival of lethally irradiated NHPs when administered at different dose levels within 1–48 hours after TBI.

<p>^A P-value by Fisher's exact test (two-tailed) for comparisons vs. vehicle groups within individual studies or in pooled group analysis</p><p>^B P-value by Log rank test (two-tailed) for comparisons vs. vehicle groups within individual studies or in pooled group analysis</p><p>^C Source I: Sichuan Atomic Energy Institute, cylindrical bundle of Co-60 rods</p><p>^D Source II: Sichuan Atomic Energy Institute, vertical array of Co-60 rods</p><p>^E Vehicle-treated animals from studies Rs-03, Rs-06, Rs-09, and Rs-14</p><p>^F Entolimod-treated animals from studies Rs-06 and Rs-14</p><p>^G Survival odds and survival odds ratios adjusted due to 100% survival are shown in italics</p><p><i>Note</i>: The frequency of moribund euthanasia was as follows: Study Rs-03–91% (1/11—found dead), Study Rs-06–100%; Study Rs-09–85% (3/20 –found dead); Rs-14–88% (1/8 –found dead). The likely cause of death in all the non-euthanized animals was acute hemorrhage.</p><p>Efficacy of a single injection of entolimod in increasing 40-day survival of lethally irradiated NHPs when administered at different dose levels within 1–48 hours after TBI.</p